Sclerotinia sclerotiorum (Ss) and Sclerotinia minor (Sm) drastically affect more than 400 plant species, and may cause crop yield reductions up to 50%. With the purpose to obtain potential microorganisms to control both pathogens, the isolation of Trichoderma strains from soil samples with different agricultural and forestry use was performed. Seventyone fungal Trichoderma isolates (IBA) were obtained through sclerotiatrap techniques. Subsequently their mycoparasitic activities on mycelium as well as on sclerotia of both phytopathogens were assessed by in vitro dual cultures. The highest percentages of colonization (>90%) of the Trichoderma strains on Ss and Sm were found for the isolates IBA3, IBA4, IBA38, IBA54. The ability of Trichoderma strains to parasitize and to degrade sclerotia in general was aggressive for the strains IBA3, IBA4, IBA23, IBA38, IBA46, IBA54, and IBA56. Based on morphological criteria, four of the most prominent strains were identified as T. viride (IBA4), T. pseudokoningii (IBA23), T. harzianum (IBA38), and T. virens (IBA46). These Trichoderma strains have important potential as antagonists to Ss and Sm.

The genus Sclerotinia produces sclerotia that remain in the soil under adverse climatic conditions for several years (Willettes and Wong, 1980; Mónaco et al., 1998; Rollan et al., 1999). The sclerotia of Ss as well as those of Sm may germinate by forming abundant mycelium; however, only Ss can germinate carpogenically by producing apothecia which carry on ascospores that represent the main source for newly initiating infection in plant tissues (Willettes and Wong, 1980; Mónaco et al., 1998). This may provide ecological advantaged to this fungus for rapidly colonizing and infecting new hosts.

During the interaction fungusfungus several complex mechanisms such as release of antibiotic compounds and lytic enzymes, hyphae attachment, and direct penetration of the host, may occur simultaneously. The mycoparasitism is based on the relationship in which one fungus obtains directly or indirectly its nutrients by invading another fungus (Gao et al., 2005). The ability of a fungus to parasite another one is partially determined by physical, chemical, or nutritional conditions (Benítez et al., 2006). Barnett and Binder (1973) classified mycoparasitism into two types: necrotrophic and biotrophic. Necrotrophic or destructive mycoparasitism typically results in death of the fungal host, and necrotrophic mycoparasites tend to be more aggressive with wide range of hosts, but without a specialized mode of action (Werner and Zadworny, 2003).

In the biotrophic mycoparasitism, the parasite development is favored by the constant contact with the host living cells. For instance, the primary antagonistic response between Trichoderma and the phytopathogen involves growth towards the susceptible hyphae, probably by positive chemotropism. Once Trichoderma detects its host, its hyphae develop a profuse branching by which the antagonist gets contact on it. Once the mycoparasite reaches the host its hyphae often roll or grow up along the pathogen mycelium (Dwivedi and Shukla, 2002).

Studies about the diversity of Trichoderma fungi for their application in integrated management of fungal diseases has received little attention in Mexico. For instance, some Trichoderma species have been successfully tested on controlling either Fusarium oxysporum or Sclerotium cepivorum under field conditions (GonzálezCárdenas et al., 2005; ÁvilaMiranda et al., 2006). Thus, the aim of this research consisted on isolating Trichoderma fungal strains from different soils of Mexico to assess their antagonistic ability towards Sclerotinia sclerotiorum and S. minor under in vitro conditions.

Materials and methods

Pathogen isolation

The pathogens were isolated and purified in the Laboratory of Soil Microbiology (Colegio de Postgraduados) from soil samples of Sclerotiniainfested plots, collected in Salamanca, Guanajuato. Sclerotinia sclerotiorum (Ss) and S. minor (Sm) were activated in potatodextroseagar (PDA, Baker®). The cultures were kept at room temperature (~20 °C) in the dark for 15 days until sclerotia production.

Isolation of Trichoderma strains

The sclerotiatrap technique was utilized to obtain antagonistic filamentous fungi to S. sclerotiorum (Ss) and S. minor (Sm). Fourteen soil samples from six states of Mexico were collected in order to get a wide range of ecosystems from which fungal strains may be isolated: 1) for Jalisco, soil was taken from the rhizosphere of mesquite vegetation (Prosopis sp.); 2) for Guanajuato, soil was collected from zones in which lettuce and garlic crops are cultivated; 3) for Oaxaca, soil samples were obtained from the rhizosphere of litchi trees, 4) for Veracruz, soil samples were collected from deciduous tree forest, 5) for Puebla, soils samples were taken from the IxtaPopo reserve whose dominant vegetation consists of pine trees, and 6) for the Estado de Mexico, soil was collected from horticultural zones of Xochimilco and Chapingo.

Small nylon bags (1cm2) were prepared in which 20 sclerotia of each pathogen were separately placed. Sclerotia were previously disinfected superficially with sodium hypochlorite at 2% for 5 min and rinsed three times with sterile distilled water. Bags were placed in duplicate in canning glass jars filled at three quarters of their capacity, at 2 cm depth in the respective soil samples. These experimental units were incubated at room temperature (~20 °C) at soil field capacity conditions. The isolation of antagonists was carried out by extracting two sclerotia at 48 h, 72 h and 96 h. Sclerotia were rinsed with sterile distilled water to eliminate excess of soil, and subsequently placed on PDAplates and incubated at room temperature (~20 °C) in the dark, then, the sclerotiaemerging antagonistic fungi of the genus Trichoderma were selected (Mónaco, 1989; Menéndez and Godeas, 1995).

In vitro confrontation of antagonists versus pathogens, and measurement of the antagonist colonization percentage on the pathogen

The pathogens as well as the isolated Trichoderma strains were confronted via dual cultures (Rollan et al., 1999). All fungi were activated in Petri dishes with PDA. Each Trichoderma strain was confronted to either Ss or Sm by triplicate, in which, an agar disk of the pathogen was placed at one side of a new petri dish, and the disk with the potential antagonist was placed at the opposite side of the plate. Petri dishes were incubated at room temperature in darkness for 15 days. Plates without fungal confrontation for each fungus were also utilized as controls. Antagonism activity was monitored by performing both daily measurements of fungal colony growth and direct observation of the plates, during 10 days. Afterwards, the colonization percentage of Trichoderma over the fungal colony of each pathogen was estimated according to the following equation (Rollan et al., 1999): C=[(DCAP/DSP)x100], where: C=colonization percentage; DCAP=distance covered by the antagonist on the pathogen colony over the axis which separates both fungi, DSP=distance between sowing points (6.5 cm). For our purposes a colonization of the Trichoderma strains higher to 70% was considered as an effective colonization over both pathogens. A completely randomized experimental design was set for each Trichoderma strain, and data were analyzed by ANOVAanalysis.

Preparation of mycoparasitized sclerotia in dual cultures

After confronting antagonists versus both pathogens, the most effective mycoparasitic Trichoderma strains were selected. Thus, 10 mL of formaldehyde 10% were added during 4 h, to each plate (Riddell, 1950). Subsequently, nine sclerotia were extracted depending on the position (first, second and third) by which they were parasitized by the Trichoderma strains. The damage of sclerotia due to the Trichoderma attack was evaluated under stereoscopic microscope as well as by performing a hardness test consisting in pressing the sclerotia with the tip of a dissection needle according to the following criteria: 1) Hard, in which sclerotia at the moment of exerting pressure with the dissection needle, their structure keep their shape and hardness; 2) Soft, in which sclerotia show little or zero resistance at pressure with dissection needle, and their structure maintain its form; 3) Hollow, where sclerotia visually maintain their structure, but at the moment of pressure, the structure gets perforated or ruined; and, 4) Out of shape (disintegrated), in which sclerotia do not show any structure, without shape and without hardness.

Results

Antagonist isolation

From the 14 soil samples, 71 isolates of filamentous fungi (IBA) were obtained and 84% of them corresponded to the genus Trichoderma, showing high competition and inhibition of sclerotia germination of Ss and Sm. The soil samples from where most of the fungal isolates were obtained corresponded to Oaxaca and Jalisco with 24 and 18 fungal isolates respectively. In contrast, the smallest numbers of fungal isolations were obtained from Veracruz with eight isolations and from the Estado de Mexico (Xochimilco and Chapingo) with seven isolates; whereas from Guanajuato only seven isolates were obtained. Antagonists from Puebla soil samples could not be isolated.

In vitro confrontation of fungal isolates and pathogens

The 71 isolated antagonists (Table 1), were classified according four categories of visual parasitism: a) Category 4, characterized by presenting 91100% of short term mycelium invasion, its accelerated growth of cottonlike mycelium on the pathogen, the formation of small heaps by which sclerotia were enveloped, thus causing sclerotiadegradation, the mycelium of the antagonist had intense green color and formed a yellowish brown halo at the meeting zone of both mycelia, b) Category 3, in which 5190% of invasion on the pathogen mycelium was observed, with small cottonlike growth and scarce formation of mycelial heaps on the sclerotia and inducing softening, the mycelium of the antagonist had intense green to olive green color and showed a brown halo only where mycelia met each other; c) Category 2, presented 1 to 50% of mycelial invasion, slow growth, not very much cottonlike mycelium on the pathogen mycelium nor the sclerotia, moreover, the antagonist strains induced the new formation of sclerotia by the pathogen, light green color of the antagonist; and d) Category 1, did not show antagonistic effect on the pathogen growth, the mycelium and sclerotia formation was similar to the control.

In general, most of the fungi showed antagonistic effects to both pathogens, but only 10 Trichoderma strains were selected on the basis of their classification as Category 4.

During the TrichodermaSm confrontation a zone of progressive inhibition was observed which may be in part attributed to the fast growth of Trichoderma. After the inhibition, a marked hyperparasitic effect of the strains IBA22, and IBA37 was detected. In addition a small orangereddish zone was observed as a result of the invasion on the pathogen's mycelium. The antagonistic ability of Trichoderma was maintained for seven days by showing a mycoparasitic effect on both pathogens. Figure 1 shows the effects of three ofthe best Trichoderma isolates (IBA4, IBA38, and IBA56) in their respective confrontation with S. sclerotiorum and S. minor.

At the fungal confrontation of TrichodermaSs, the pathogen limited the invasion of the antagonist by elevating its mycelium and then, acting as a barrier. At the contact with the antagonistic fungal strain, Sclerotinia stopped its growth and started the formation of sclerotia on the periphery of its fungal colony (Figure 1). The sclerotia development was irregular since few of them had large size or deformations (data nonpresented). Twentyfive Trichoderma strains completely invaded either the colony or the sclerotia of the pathogen. The average period of colonization of the antagonistic over the pathogen was between 810 days.

Measurement of the antagonist colonization percentage

There were observed significant differences (P<0.001) on the colonization percentage of the Trichoderma strains on the fungal pathogens, and only ten strains were preselected according to the best colonization percentages (IBA1, IBA3, IBA4, IBA22, IBA23, IBA37, IBA38, IBA46, IBA54, and IBA56). Figure 2 shows the colonization of 10 Trichoderma strains on the mycelium of Ss and Sm. The colonization percentage of IBA54 on the mycelium of both pathogens was higher than 90%, which was obtained in less time (67 days) in comparison to the strains IBA3, IBA4, and IBA38 (Figure 2). These four Trichoderma strains had excellent abilities for invading the mycelium of both pathogens, by which these strains were selected for the next experimental stages.

Mycoparasitism of sclerotia in dual cultures

The zone of attack induced on the pathogen by Trichoderma strains, increased with the time, and it was accompanied by the destruction of the pathogen mycelium. In addition, the observed mycoparasitism of sclerotia resulted on severe damage to their structure. Some sclerotia structures were hollow, soft or disintegrated by the activity of the 10 selected strains, likewise, variations in sclerotia size were observed (Figures 3 and 4). Table 2 shows the degree of sclerotia parasitism after 7 days of the contact between the antagonists with the pathogen. Significant differences in sclerotia degradation were observed (Tukey, c=0.05) at the moment of its invasion for the antagonists.

Main damages caused by the IBA4 strain to Sssclerotia consisted on deformations, collapsing, and cracking (Figure 3 b1b3) when compared to the control (Figure 3 a1a3); the strain IBA23 produced collapsing, distortion, and increase in size (Figure 3 c1c3); while the strain IBA56 besides affecting and diminishing sclerotia size also produced their collapsing (Figure 3 e1e3). In contrast, the strain IBA22 did not produce internal damage of sclerotia, but did Figure 3. Mycoparasitism of sclerotia of Sclerotinia sclerotiorum by live Trichoderma strains, where: a) Control, b) IBA4, c) IBA23, d) IBA38, e) IBA56, and f) IBA22. Numerals (1 3) refer to the position of sclerotia (1=first, 2=second, and 3=third position) at the moment of meeting with the antagonist in dual cultures, after completely invading the colony of S. sclerotiorum. Line = 1 mm. produce distortion in size, inducing larger sclerotia than the control (Figure 3 f1f3).

The damages observed for Smsclerotia due to three strains (IBA4, IBA23, and IBA38) were more remarkable since they caused disintegration of sclerotia (Figure 4 d1d3), distortion, and collapsing (Figure 4 b1b2, c1c3, d1d3). Although the strain IBA22 did not induced damage on sclerotia, it caused structure distortion (Figure 4 e1e3) in comparison to the control (Figure 4 a1a3). The most severe damages on the sclerotia structures corresponded to those sclerotia that were firstly reached by the antagonist.

Based on morphological criteria (Bissett, 1984, 1991), four of the most prominent strains were identified as T. viride (IBA4), T. pseudokoningii (IBA23), T. harzianum (IBA38), and T. virens (IBA46), have important potential as antagonists to Ss and Sm.

Discussion

The in vitro assay confirmed the effectiveness of Trichoderma isolates for controlling the growth of Ss and Sm. The Trichoderma isolates showed clear antagonistic effect by significantly inhibiting the growth of both pathogens. Similar results have been reported for Trichoderma harzianum isolates against Phytophthora capsici in dual cultures (Ezziyyani et al., 2004). The mycoparasitism of the Trichoderma strains varied according to its combination with the pathogen species. The inhibition zone produced by some Trichoderma strains when facing the pathogen increased with the time and resulted in mycelium and sclerotia destruction. The strain IBA23 reduced the sclerotia formation of Sm, and this effect agrees with that described by Molina et al. (2006). The mycoparasitic effect of the Trichoderma strains on sclerotia has ecological implications since not only their structure was destroyed but also this fungal attack may significantly prevent sclerotia germination.

All Trichoderma strains were capable of growing and sporulating on the pathogen sclerotia, although not all sclerotia were fully parasitized. The latest effect agrees with results from Rollan et al. (1999) in which some Trichoderma species are able to parasite sclerotia, but their effectiveness depended on the target pathogen.

The growth inhibition observed on the fungal colony of both pathogens by the antagonists may be attributed to the release of hydrolytic enzymes such as β, 13glucanase, chitinase, protease, and cellulase (Ezziyyani et al., 2004), which are a key step for initiating the pathogen cell wall degradation during mycoparasitism (Rey et al., 2000). The lysis induced by these enzymatic activities depends on the melanin content of the host, and the melanization is directly related to the fungal development and volatile metabolites (Dal et al., 1997; Brozóvá, 2004).

Sclerotia formation has an ecological importance due to the fact that it guarantees pathogen persistence in soil. The observed mycoparasitism on sclerotia has special relevance since the sclerotia formation and persistence may be significantly reduced by the presence of an effective fungal hyperparasite. Our results about sclerotia degradation agree with some experimental reports. GarcíaGarza et al. (1997) described the degradation of sclerotia by Trichoderma hamatum, which initiated its attack in the melanized layer and then invading, and causing lysis of the inner structure of the sclerotia which had either constrained or hollow appearance. These processes were observed for the strains IBA23 and IBA38 on S. minor, whose sclerotia show hollows and/or cracks. Bolton et al. (2006) described that the inner structure of sclerotia has a fibroid matrix consisting of carbohydrates, mainly βglucan and proteins; therefore, the internal degradation of sclerotia by antagonists may be partly due to βglucanase releasing. This may justify in part, why the internal sclerotia degradation is the first process during the antagonist attack, and subsequently the structure of the sclerotia collapses. The elimination of the sclerotia matrix increases the opportunity of being also parasitized by other organisms. GarcíaGarza et al. (1997) suggest that sclerotia under field conditions may have an infected surface, allowing the easy penetration by other organisms, and then resulting on the degradation of these persistence structures. The fungal cell wall is a complex structure made up of different polymers that define their degradation or survival. Generally, the cell wall is constituted by an internal layer of crystalline mycofibrils wrapped up in an amorphous matrix, together with one or more external layers. The most abundant components of fungal cell walls are chitin, β1,3glucane and glycoproteins, which confer them special characteristics. There is evidence that some nanoproteins are bounded with β1,3glucane and chitin fibers, forming a very resistant net (Mohammed et al., 2004); that is why the secretion of lytic enzymes such as glucanase and chitinase by Trichoderma make the mycoparasitism be more efficient.

Even though it is true that under field conditions there are other biotic or abiotic factors that may influence the expression of the antagonism, the in vitro assays allow the selection of Trichoderma species suitable as biocontrol agents, but further research is needed to validate the most prominent strains under systems that resemble natural conditions or under field conditions. It may be concluded that it is possible to isolate antagonistic fungi from several soils with different use such as forestry and agricultural. In addition, Trichoderma strains are appropriate agents for controlling the growth and sclerotia of either Ss or Sm.